I have expressed GST-tagged protein in BL21(DE3)pLysS cells but I am not able to get the protein in soluble form. Most of the protein is in inclusion body. The induction conditions used are 1mM IPTG at 30ºC for 6h at 220 rpm. please suggest me conditions to adopt so that the protein is in soluble form as I have to use it for GST-pull down.
The first and easiest thing to try is lowering the temperature during induction (as low as 16oC). By reducing the rate of protein production, this sometimes results in a higher proportion of soluble protein.
The first and easiest thing to try is lowering the temperature during induction (as low as 16oC). By reducing the rate of protein production, this sometimes results in a higher proportion of soluble protein.
If it is an E.coli or protein from any other prokaryote, you have high chance of getting it successful if you induce them at low temperature(over night) as suggested by Adam(I would test different IPTG concentration in a pilot scale). I got enough human protein GST tagged by expressing E.coli(Rosetta DE3).
Lysis and buffer conditions make to yield better during purification.
To avoid the inclusion bodies you grow the cells at 37 degree until it reach the OD-0.4 and then induce with 0.5 mM IPTG and keep the cells at 18 degree over night .
I hope it will help you i am using the same condition to get my protein in soluble condition.
low the inducer concentration and the expression temperature (as Adam mentioned). Meanwhile I am not is it possible for you to attach another fusion tags such as maltose binding protein which normally can help the solubility of the fusion protein, like GST-MBP-TP or MBP-GST-TP. Some strange strains which has the cell chaperon for protein folding and forming of the disulfide bonds
Steven
All the suggestions mentioned by others may work. i.e.. lowering the temp. lowering the Inucer conc. Sometime lowering the time of induction also works. If nothing works you can still purify you protein of Interest In denature form from Inclusion bodies and then reture it by dialysis.
Hi Sonalika,
what kind of protein did you fuse to GST? Membran proteins tend to be a little bit more tricky than others.
Personally I am working on carotenoid cleavage proteins and we yielded good results when expressing these fusion proteins together with the Gro EL-ES chaperone system, which assists correct folding. So if you have a membran (associated) protein my first suggestion would be to change the expression strain to DE3 Gro7 or Rosetta Gro7.
As the others stated before induction point and duration is important. Try initial growth to OD of 0.5 at 37°C than induce with 1mM Iptg and lower temperature to 28°C for 4-6h and harvest. Expression at 18°C over night never yielded good results in our lab. As another optimization try induction with 0.1, 0.5M and max concentration of IPTG and see if you have huge differences. If not choose a lower concentration of IPTG for induction to reduce stress of expression strain.
All the best for your experiments
Mark
i tried induction at 18ºC with 0.5mM IPTG overnight but it didn't work, protein is till in the inclusion body. its not a memrane protein though it contains a memebrane amphipathic helix
To get the protein in soluble form you can add 0.1% Triton ™X-100 and 0.3% sarkosyl during the lysis steps.
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